Over a decade ago, we demonstrated that the latent viral reservoir in the resting CD4+ T cell compartment persists in virtually all HIV-infected individuals receiving clinically effective ART. Consequently, this viral reservoir has become a major impediment to the eradication of HIV in infected individuals receiving ART. During the past year, we have focused our research on: 1) delineating the role of CpG methylation of HIV DNA in the maintenance of HIV latency in infected individuals receiving ART and 2) examining the effect of HDACis on the degree and extent of HIV expression in latently infected, resting CD4+ T cells in infected individuals receiving ART. First, we investigated the role of CpG methylation of HIV proviral DNA in the maintenance of the latent viral reservoir in infected individuals receiving ART. CpG methylation has been implicated in suppressing the expression of host genes and various endogenous and/or infectious retroviruses in the past. In recent years, it has been suggested that the degree of methylation in the promoter/enhancer region, 5 long terminal repeat (LTR), of HIV DNA inversely correlates with the level of viral expression following stimulation of chronically infected cell lines and other in vitro infection models. However, studies addressing the level of methylation in the 5LTR of HIV DNA in resting CD4+ T cells of aviremic individuals receiving ART have been lacking. Using DNA isolated from resting CD4+ T cells of 11 infected individuals receiving ART, we demonstrated that the median frequency of methylated CpG dinucleotides within the HIV 5 LTR was only 2.4%. The level of HIV 5 LTR methylation in activated CD4+ T cells was similar (median 2.5%) to that of resting CD4+ T cells. Our data suggest that HIV DNA carrying high levels of 5 LTR methylation is rare in latently infected, resting CD4+ T cells of aviremic infected individuals. Thus, the latent viral reservoir is likely maintained predominantly by mechanisms other than methylation of HIV 5 LTR. Second, we examined the effect of HDACis on the degree and extent of HIV expression in latently infected, resting CD4+ T cells in infected individuals receiving ART. We have previously demonstrated that intermittent administration of the cellular activating agent interleukin (IL)-2 could reduce the frequency of infectious HIV in the latent viral reservoir in patients receiving ART. However, administration of IL-2 or anti-CD3 antibody did not lead to eradication of HIV in infected individuals receiving ART. In recent years, valproic acid (VPA), an HDACi, has been tested in humans as a potential virus purging agent and was shown in one study to diminish the size of the latent viral reservoir in infected individuals receiving ART. However, subsequent studies have demonstrated no appreciable reduction of the latent viral reservoir following treatment with VPA. Given these conflicting results and considering that a major thrust of HIV therapeutic research at the present time is the development of strategies aimed at eradicating the virus, it is of considerable interest and importance to carefully evaluate the capacity of HDACis to induce HIV expression in the latent viral reservoir. It has been suggested that such HIV purging agents may decrease the viral burden by inducing virus expression leading to virus-induced cytopathic effects. We measured the copy number of virion-associated HIV RNA in the culture supernatants of cells following incubation with media alone, a variety of HDACis, or T cell activators. In the majority of HIV-aviremic individuals receiving ART, HIV RNA was not detected when cells were incubated with media alone, VPA, suberoylanilide hydroxamic acid (SAHA), or oxamflatin. However, substantially higher levels of HIV RNA were detected when resting CD4+ T cells were incubated with prostratin or anti-CD3 antibody. The level of HIV RNA in the culture supernatant when cells were incubated with media alone was not statistically different when compared to that of cells incubated with VPA, SAHA, or oxamflatin. In contrast, prostratin and anti-CD3 antibody induced significantly higher levels of virions relative to that of cells incubated with media. We then investigated the degree of heterogeneity of virion-associated HIV RNA in the culture supernatants upon incubation of resting CD4+ T cells with HDACis versus T cell activating reagents. Using heteroduplex mobility assays, we demonstrated that the sequences of HIV envelope recovered from cells that had been incubated with prostratin and anti-CD3 antibody showed high levels of heterogeneity, likely a result of maximal stimulation of HIV expression in cells carrying inducible virus. In contrast, the sequences of HIV envelope originating from the resting CD4+ T cell culture stimulated with HDACis showed relatively low degrees of heterogeneity, suggesting that HDACis may only stimulate HIV expression in a small fraction of latently infected, resting CD4+ T cells. Our data suggest that HDACis would likely be ineffective at eliminating the latent viral reservoir in infected individuals receiving ART and other therapeutic strategies may be needed to eliminate latently infected, resting CD4+ T cells in vivo.